Nucleotide sequences which code for the RPSL gene

ABSTRACT

The present invention relates to polynucleotides corresponding to the rpsL gene and which encode ribosomal protein S12, methods of producing L-amino acids, and methods of screening for polynucleotides which encode proteins having ribosomal protein S12 activity.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] The presen application claims priority to German Application No.DE 10107230.9 filed Feb. 16, 2001, the entire contents of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention provides nucleotide sequences from coryneformbacteria which code for the rpsL gene and a process for the fermentativepreparation of amino acids using bacteria in which the rpsL gene isenhanced.

[0004] 2. Discussion of the Background

[0005] L-Amino acids, in particular L-lysine, are used in human medicineand in the pharmaceuticals industry, in the foodstuffs industry and veryparticularly in animal nutrition.

[0006] It is known that amino acids are prepared by fermentation fromstrains of Coryneform bacteria, in particular Corynebacteriumglutamicum. Because of their great importance, work is constantly beingundertaken to improve the preparation processes. Improvements to theprocess can relate to fermentation measures, such as, for example,stirring and supply of oxygen, or the composition of the nutrient media,such as, for example, the sugar concentration during the fermentation,or the working up to the product form by, for example, ion exchangechromatography, or the intrinsic output properties of the microorganismitself.

[0007] Methods of mutagenesis, selection and mutant selection are usedto improve the output properties of these microorganisms. Strains whichare resistant to antimetabolites or are auxotrophic for metabolites ofregulatory importance and produce amino acids are obtained in thismanner.

[0008] Methods of the recombinant DNA technique have also been employedfor some years for improving the strain of Corynebacterium strains whichproduce L-amino acid, by amplifying individual amino acid biosynthesisgenes and investigating the effect on the amino acid production.

[0009] However, there remains a critical need for improved methods ofproducing L-amino acids and thus for the provision of strains ofbacteria producing higher amounts of L-amino acids. On a commercial orindustrial scale even small improvements in the yield of L-amino acids,or the efficiency of their production, are economically significant.Prior to the present invention, it was not recognized that enhancementor over-expression of rpsL gene encoding the ribosomal protein S12 wouldimprove L-amino acid yields.

SUMMARY OF THE INVENTION

[0010] One object of the present invention, is providing a new processadjuvant for improving the fermentative production of L-amino acids,particularly L-lysine and L-glutamate. Such process adjuvants includeenhanced bacteria, preferably enhanced Coryneform bacteria which expresshigh amounts of ribosomal protein S12 which is encoded by the rpsL gene.

[0011] Thus, another object of the present invention is providing suchan enhanced bacterium, which expresses an enhanced amount of ribosomalprotein S12 or gene products of the rpsL gene.

[0012] Another object of the present invention is providing a bacterium,preferably a Coryneform bacterium, which expresses a polypeptide thathas an enhanced ribosomal protein S12 activity. In a preferredembodiment, the gene encoding the enhance ribosomal protein S12comprises the sequence of SEQ ID NO: 3

[0013] Another object of the present invention is an enhanced ribosomalprotein S12 which comprises the amino sequence of SEQ ID NO: 4.

[0014] Another object of the invention is to provide a nucleotidesequence encoding a polypeptide which has ribosomal protein S12sequence. One embodiment of such a sequence is the nucleotide sequenceof SEQ ID NO: 1.

[0015] A further object of the invention is a method of making ribosomalprotein S12 or an isolated polypeptide having a ribosomal protein S12activity, as well as use of such isolated polypeptides in the productionof amino acids. One embodiment of such a polypeptide is the polypeptidehaving the amino acid sequence of SEQ ID NO: 2.

[0016] Other objects of the invention include methods of detectingnucleic acid sequences homologous to SEQ ID NO: 1, particularly nucleicacid sequences encoding polypeptides that have ribosomal protein S12activity, and methods of making nucleic acids encoding suchpolypeptides.

[0017] The above objects highlight certain aspects of the invention.Additional objects, aspects and embodiments of the invention are foundin the following detailed description of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0018] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art of molecular biology. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described herein. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and are notintended to be limiting.

[0019] Reference is made to standard textbooks of molecular biology thatcontain definitions and methods and means for carrying out basictechniques, encompassed by the present invention. See, for example,Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1982) and Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York(1989) and the various references cited therein.

[0020] Where L-amino acids or amino acids are mentioned in thefollowing, this means one or more amino acid, including their salts,chosen from the group consisting of L-asparagine, L-threonine, L-serine,L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine,L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine,L-lysine, L-tryptophan and L-arginine. L-Lysine is particularlypreferred.

[0021] When L-lysine or lysine are mentioned in the following, not onlythe bases but also the salts, such as e.g. lysine monohydrochloride orlysine sulfate, are meant by this.

[0022] The invention provides an isolated polynucleotide from Coryneformbacteria, comprising a polynucleotide sequence which codes for the rpsLgene chosen from the group consisting of

[0023] a) polynucleotide which is identical to the extent of at least70% to a polynucleotide which codes for a polypeptide which comprisesthe amino acid sequence of SEQ ID No. 2,

[0024] b) polynucleotide which codes for a polypeptide which comprisesan amino acid sequence which is identical to the extent of at least 70%to the amino acid sequence of SEQ ID No. 2,

[0025] c) polynucleotide which is complementary to the polynucleotidesof a) or b), and

[0026] d) polynucleotide comprising at least 15 successive nucleotidesof the polynucleotide sequence of a), b) or c),

[0027] the polypeptide preferably having the activity of the ribosomalprotein S12.

[0028] The invention also provides the abovementioned polynucleotide,this preferably being a DNA which is capable of replication, comprising:

[0029] (i) the nucleotide sequence shown in SEQ ID no. 1, or

[0030] (ii) at least one sequence which corresponds to sequence (i)within the range of the degeneration of the genetic code, or

[0031] (iii) at least one sequence which hybridizes with the sequencecomplementary to sequence (i) or (ii), and optionally

[0032] (iv) sense mutations of neutral function in (i) which do notmodify the activity of the protein/polypeptide

[0033] Finally, the invention also provides polynucleotides chosen fromthe group consisting of

[0034] a) polynucleotides comprising at least 15 successive nucleotideschosen from the nucleotide sequence of SEQ ID No. 1 between positions 1and 499

[0035] b) polynucleotides comprising at least 15 successive nucleotideschosen from the nucleotide sequence of SEQ ID No. 1 between positions500 and 883

[0036] c) polynucleotides comprising at least 15 successive nucleotideschosen from the nucleotide sequence of SEQ ID No. 1 between positions884 and 1775.

[0037] The invention also provides

[0038] a polynucleotide, in particular DNA, which is capable ofreplication and comprises the nucleotide sequence as shown in SEQ ID No.1;

[0039] a polynucleotide which codes for a polypeptide which comprisesthe amino acid sequence as shown in SEQ ID No. 2;

[0040] a vector containing the polynucleotide according to theinvention, in particular a shuttle vector or plasmid vector, and

[0041] Coryneform bacteria which contain the vector or in which the rpsLgene is enhanced.

[0042] The invention also provides polynucleotides which substantiallycomprise a polynucleotide sequence, which are obtainable by screening bymeans of hybridization of a corresponding gene library of a Coryneformbacterium, which comprises the complete gene or parts thereof, with aprobe which comprises the sequence of the polynucleotide according tothe invention according to SEQ ID No. 1 or a fragment thereof, andisolation of the polynucleotide sequence mentioned.

[0043] Polynucleotides which comprise the sequences according to theinvention are suitable as hybridization probes for RNA, cDNA and DNA, inorder to isolate, in the full length, nucleic acids or polynucleotidesor genes which code for the ribosomal protein S12 or to isolate thosenucleic acids or polynucleotides or genes which have a high similaritywith the sequence of the rpsL gene. They are also suitable forincorporation into so-called “arrays”, micro arrays” or “DNA chips” inorder to detect and determine the corresponding polynucleotides [sic]

[0044] Polynucleotides which comprise the sequences according to theinvention are furthermore suitable as primers with the aid of which DNAof genes which code for the ribosomal protein S12 can be prepared by thepolymerase chain reaction (PCR).

[0045] Such oligonucleotides which serve as probes or primers compriseat least 25, 26, 27, 28, 29 or 30, preferably at least 20, 21, 22, 23 or24, very particularly preferably at least 15, 16, 17, 18 or 19successive nucleotides. Oligonucleotides with a length of at least 31,32, 33, 34, 35, 36, 37, 38, 39 or 40, or at least 41, 42, 43, 44, 45,46, 47, 48, 49 or 50 nucleotides are also suitable. oligonucleotideswith a length of at least 100, 150, 200, 250 or 300 nucleotides areoptionally also suitable.

[0046] “Isolated” means separated out of its natural environment.

[0047] “Polynucleotide” in general relates to polyribonucleotides andpolydeoxyribonucleotides, it being possible for these to be non-modifiedRNA or DNA or modified RNA or DNA.

[0048] The polynucleotides according to the invention include apolynucleotide according to SEQ ID No. 1 or a fragment preparedtherefrom and also those which are at least in particular 70% to 80%,preferably at least 81% to 85%, particularly preferably at least 86% to90%, and very particularly preferably at least 91%, 93%, 95%, 97% or 99%identical to the polynucleotide according to SEQ ID No. 1 or a fragmentprepared therefrom.

[0049] “Polypeptides” are understood as meaning peptides or proteinswhich comprise two or more amino acids bonded via peptide bonds.

[0050] The polypeptides according to the invention include a polypeptideaccording to SEQ ID No. 2, in particular those with the biologicalactivity of the ribosomal protein S12 and also those which are at least70% to 80%, preferably at least 81% to 85%, particularly preferably atleast 86% to 90%, and very particularly preferably at least 91%, 93%,95%, 97% or 99% identical to the polypeptide according to SEQ ID No. 2and have the activity mentioned.

[0051] The invention furthermore relates to a process for thefermermentative [sic] preparation of amino acids chosen from the groupconsisting of L-asparagine, L-threonine, L-serine, L-glutamate,L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine,L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine,L-tryptophan and L-arginine using Coryneform bacteria which inparticular already produce amino acids and in which the nucleotidesequences which code for the rpsL gene are enhanced, in particularover-expressed.

[0052] The term “enhancement” in this connection describes the increasein the intracellular activity of one or more enzymes or proteins in amicroorganism which are coded by the corresponding DNA, for example byincreasing the number of copies of the gene or genes, using a potentpromoter or using a gene or allele which codes for a correspondingenzyme or protein with a high activity, and optionally combining thesemeasures.

[0053] The microorganisms which the present invention provides canproduce L-amino acids from glucose, sucrose, lactose, fructose, maltose,molasses, starch, cellulose or from glycerol and ethanol. They can berepresentatives of Coryneform bacteria, in particular of the genusCorynebacterium. Of the genus Corynebacterium, there may be mentioned inparticular the species Corynebacterium glutamicum, which is known amongexperts for its ability to produce L-amino acids.

[0054] Suitable strains of the genus Corynebacterium, in particular ofthe species Corynebacterium glutamicum (C. glutamicum), are inparticular the known wild-type strains

[0055]Corynebacterium glutamicum ATCC13032

[0056]Corynebacterium acetoglutamicum ATCC15806

[0057]Corynebacterium acetoacidophilum ATCC13870

[0058]Corynebacterium thermoaminogenes FERM BP-1539

[0059]Corynebacterium melassecola ATCC17965

[0060]Brevibacterium flavum ATCC14067

[0061]Brevibacterium lactofermentum ATCC13869 and

[0062]Brevibacterium divaricatum ATCC14020

[0063] and L-amino acid-producing mutants or strains prepared therefrom,such as, for example, the L-lysine-producing strains

[0064]Corynebacterium glutamicum FERM-P 1709

[0065]Brevibacterium flavum FERM-P 1708

[0066]Brevibacterium lactofermentum FERM-P 1712

[0067]Corynebacterium glutamicum FERM-P 6463

[0068]Corynebacterium glutamicum FERM-P 6464

[0069]Corynebacterium glutamicum DM58-1

[0070]Corynebacterium glutamicum DG52-5

[0071]Corynebacterium glutamicum DSM5714 and

[0072]Corynebacterium glutamicum DSM12866.

[0073] Preferably, a bacterial strain enhanced for expression of a rpsLgene that encodes a polypeptide with ribosomal protein S12 activity willimprove amino acid yield at least 1%.

[0074] The new rpsL gene from C. glutamicum which codes for theribosomal protein S12 has been isolated.

[0075] To isolate the rpsL gene or also other genes of C. glutamicum, agene library of this microorganism is first set up in Escherichia coli(E. coli). The setting up of gene libraries is described in generallyknown textbooks and handbooks. The textbook by Winnacker: Gene undKlone, Eine Einführung in die Gentechnologie [Genes and Clones, AnIntroduction to Genetic Engineering] (Verlag Chemie, Weinheim, Germany,1990), or the handbook by Sambrook et al.: Molecular Cloning, ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1989) may bementioned as an example. A well-known gene library is that of the E.coli K-12 strain W3110 set up in λ vectors by Kohara et al. (Cell 50,495-508 (1987)). Bathe et al. (Molecular and General Genetics,252:255-265, 1996) describe a gene library of C. glutamicum ATCC13032,which was set up with the aid of the cosmid vector SuperCos I (Wahl etal., 1987, Proceedings of the National Academy of Sciences USA,84:2160-2164) in the E. coli K-12 strain NM554 (Raleigh et al., 1988,Nucleic Acids Research 16:1563-1575). Börmann et al. (MolecularMicrobiology 6(3), 317-326) (1992)) in turn describe a gene library ofC. glutamicum ATCC13032 using the cosmid pHC79 (Hohn and Collins, Gene11, 291-298 (1980)).

[0076] To prepare a gene library of C. glutamicum in E. coli it is alsopossible to use plasmids such as pBR322 (Bolivar, Life Sciences, 25,807-818 (1979)) or pUC9 (Vieira et al., 1982, Gene, 19:259-268).Suitable hosts are, in particular, those E. coli strains which arerestriction- and recombination-defective. An example of these is thestrain DH5αmcr, which has been described by Grant et al. (Proceedings ofthe National Academy of Sciences USA, 87 (1990) 4645-4649). The long DNAfragments cloned with the aid of cosmids can in turn be subcloned in theusual vectors suitable for sequencing and then sequenced, as isdescribed e.g. by Sanger et al. (Proceedings of the National Academy ofSciences of the United States of America, 74:5463-5467, 1977).

[0077] The resulting DNA sequences can then be investigated with knownalgorithms or sequence analysis programs, such as e.g. that of Staden(Nucleic Acids Research 14, 217-232(1986)), that of Marck (Nucleic AcidsResearch 16, 1829-1836 (1988)) or the GCG program of Butler (Methods ofBiochemical Analysis 39, 74-97 (1998)).

[0078] The new DNA sequence of C. glutamicum which codes for the rpsLgene and which, as SEQ ID No. 1, is a constituent of the presentinvention has been found. The amino acid sequence of the correspondingprotein has furthermore been derived from the present DNA sequence bythe methods described above. The resulting amino acid sequence of therpsL gene product is shown in SEQ ID No. 2. It is known that enzymesendogenous in the host can split off the N-terminal amino acidmethionine or formylmethionine of the protein formed.

[0079] Coding DNA sequences which result from SEQ ID No. 1 by thedegeneracy of the genetic code are also a constituent of the invention.In the same way, DNA sequences which hybridize with SEQ ID No. 1 orparts of SEQ ID No. 1 are a constituent of the invention. Conservativeamino acid exchanges, such as e.g. exchange of glycine for alanine or ofaspartic acid for glutamic acid in proteins, are furthermore known amongexperts as “sense mutations” which do not lead to a fundamental changein the activity of the protein, i.e. are of neutral function. Suchmutations are also called, inter alia, neutral substitutions. It isfurthermore known that changes on the N and/or C terminus of a proteincannot substantially impair or can even stabilize the function thereof.Information in this context can be found by the expert, inter alia, inBen-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)), inO'Regan et al. (Gene 77:237-251 (1989)), in Sahin-Toth et al. (ProteinSciences 3:240-247 (1994)), in Hochuli et al. (Bio/Technology6:1321-1325 (1988)) and in known textbooks of genetics and molecularbiology. Amino acid sequences which result in a corresponding mannerfrom SEQ ID No. 2 are also a constituent of the invention.

[0080] In the same way, DNA sequences which hybridize with SEQ ID No. 1or parts of SEQ ID No. 1 are a constituent of the invention. Finally,DNA sequences which are prepared by the polymerase chain reaction (PCR)using primers which result from SEQ ID No. 1 are a constituent of theinvention. Such oligonucleotides typically have a length of at least 15nucleotides.

[0081] Instructions for identifying DNA sequences by means ofhybridization can be found by the expert, inter alia, in the handbook“The DIG System Users Guide for Filter Hybridization” from BoehringerMannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al.(International Journal of Systematic Bacteriology (1991) 41: 255-260).The hybridization takes place under stringent conditions, that is to sayonly hybrids in which the probe and target sequence, i. e. thepolynucleotides treated with the probe, are at least 70% identical areformed. It is known that the stringency of the hybridization, includingthe washing steps, is influenced or determined by varying the buffercomposition, the temperature and the salt concentration. Thehybridization reaction is preferably carried out under a relatively lowstringency compared with the washing steps (Hybaid Hybridisation Guide,Hybaid Limited, Teddington, UK, 1996).

[0082] A 5× SSC buffer at a temperature of approx. 50° C.-68° C., forexample, can be employed for the hybridization reaction. Probes can alsohybridize here with polynucleotides which are less than 70% identical tothe sequence of the probe. Such hybrids are less stable and are removedby washing under stringent conditions. This can be achieved, forexample, by lowering the salt concentration to 2× SSC and optionallysubsequently 0.5× SSC (The DIG System User's Guide for FilterHybridisation, Boehringer Mannheim, Mannheim, Germany, 1995) atemperature of approx. 50° C.-68° C. being established. It is optionallypossible to lower the salt concentration to 0.1× SSC. Polynucleotidefragments which are, for example, at least 70% or at least 80% or atleast 90% to 95% identical to the sequence of the probe employed can beisolated by increasing the hybridization temperature stepwise from 50°C. to 68° C. in steps of approx. 1-2° C. Further instructions onhybridization are obtainable on the market in the form of so-called kits(e.g. DIG Easy Hyb from Roche Diagnostics GmbH, Mannheim, Germany,Catalogue No. 1603558).

[0083] Instructions for amplification of DNA sequences with the aid ofthe polymerase chain reaction (PCR) can be found by the expert, interalia, in the handbook by Gait: Oligonukleotide [sic] synthesis: APractical Approach (IRL Press, Oxford, UK, 1984) and in Newton andGraham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).

[0084] It has been found that Coryneform bacteria produce amino acids inan improved manner after enhancement of the rpsL gene.

[0085] To achieve an over-expression, the number of copies of thecorresponding genes can be increased, or the promoter and regulationregion or the ribosome binding site upstream of the structural gene canbe mutated. Expression cassettes which are incorporated upstream of thestructural gene act in the same way. By inducible promoters, it isadditionally possible to increase the expression in the course offermentative amino acid production. The expression is likewise improvedby measures to prolong the life of the m-RNA. Furthermore, the enzymeactivity is also increased by preventing the degradation of the enzymeprotein. The genes or gene constructs can either be present in plasmidswith a varying number of copies, or can be integrated and amplified inthe chromosome. Alternatively, an over-expression of the genes inquestion can furthermore be achieved by changing the composition of themedia and the culture procedure.

[0086] Instructions in this context can be found by the expert, interalia, in Martin et al. (Bio/Technology 5, 137-146 (1987)), in Guerreroet al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology6, 428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), inEuropean Patent Specification 0 472 869, in U.S. Pat. No. 4,601,893, inSchwarzer and Pühler (Bio/Technology 9, 84-87 (1991), in Reinscheid etal. (Applied and Environmental Microbiology 60, 126-132 (1994)), inLaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), inPatent Application WO 96/15246, in Malumbres et al. (Gene 134, 15-24(1993)), in Japanese Laid-Open Specification JP-A-10-229891, in Jensenand Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), inMakrides (Microbiological Reviews 60:512-538 (1996)) and in knowntextbooks of genetics and molecular biology.

[0087] By way of example, for enhancement the rpsL gene according to theinvention was over-expressed with the aid of episomal plasmids. Suitableplasmids are those which are replicated in Coryneform bacteria. Numerousknown plasmid vectors, such as e.g. pZ1 (Menkel et al., Applied andEnvironmental Microbiology (1989) 64: 549-554), pEKEx1 (Eikmanns et al.,Gene 102:93-98 (1991)) or pHS2-1 (Sonnen et al., Gene 107:69-74 (1991))are based on the cryptic plasmids pHM1519, pBL1 or pGA1. Other plasmidvectors, such as e.g. those based on pCG4 (U.S. Pat. No. 4,489,160), orpNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124(1990)), or pAG1 (U.S. Pat. No. 5,158,891), can be used in the samemanner.

[0088] Plasmid vectors which are furthermore suitable are also thosewith the aid of which the process of gene amplification by integrationinto the chromosome can be used, as has been described, for example, byReinscheid et al. (Applied and Environmental Microbiology 60, 126-132(1994)) for duplication or amplification of the hom-thrB operon. In thismethod, the complete gene is cloned in a plasmid vector which canreplicate in a host (typically E. coli), but not in C. glutamicum.Possible vectors are, for example, pSUP301 (Simon et al., Bio/Technology1, 784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73(1994)), pGEM-T (Promega corporation, Madison, Wis., USA), pCR2.1-TOPO(Shuman (1994). Journal of Biological Chemistry 25 269:32678-84; U.S.Pat. No. 5,487,993), pCR®Blunt (Invitrogen, Groningen, Holland; Bernardet al., Journal of Molecular Biology, 234: 534-541 (1993)), pEM1(Schrumpf et al, 1991, Journal of Bacteriology 173:4510-4516) or pBGS8(Spratt et al., 1986, Gene 41: 337-342). The plasmid vector whichcontains the gene to be amplified is then transferred into the desiredstrain of C. glutamicum by conjugation or transformation. The method ofconjugation is described, for example, by Schäfer et al. (Applied andEnvironmental Microbiology 60, 756-759 (1994)). Methods fortransformation are described, for example, by Thierbach et al. (AppliedMicrobiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan(Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMSMicrobiological Letters 123, 343-347 (1994)). After homologousrecombination by means of a “cross over” event, the resulting straincontains at least two copies of the gene in question.

[0089] It has furthermore been found that amino acid exchanges in thesection between position 38 to 48 of the amino acid sequence of theribosomal protein S12 shown in SEQ ID No. 2 improve the lysineproduction of Coryneform bacteria.

[0090] Preferably, L-lysine at position 43 is exchanged for any otherproteinogenic amino acid excluding L-lysine, exchange for L-histidine orL-arginine being preferred. Exchange for L-arginine is very particularlypreferred.

[0091] The base sequence of the allele rpsL-1545 contained in strainDM1545 is shown in SEQ ID No. 3. The rpsL-1545 allele codes for aprotein, the amino acid sequence of which is shown in SEQ ID No. 4. Theprotein contains L-arginine at position 43. The DNA sequence of therpsL-1545 allele (SEQ ID No. 3) contains the base guanine instead of thebase adenine contained at position 627 in the rpsL wild-type gene (SEQID No. 1).

[0092] For mutagenesis, conventional mutagenesis processes can be used,using mutagenic substances such as, for example,N-methyl-N′-nitro-N-nitrosoguanidine or ultraviolet light. In vitromethods, such as, for example, a treatment with hydroxylamine (Miller,J. H.: A Short Course in Bacterial Genetics. A Laboratory Manual andHandbook for Escherichia coli and Related Bacteria, Cold Spring HarborLaboratory Press, Cold Spring Harbor, 1992) or mutagenicoligonucleotides (T. A. Brown: Gentechnologie für Einsteiger [GeneticEngineering for Beginners], Spektrum Akademischer Verlag, Heidelberg,1993) or the polymerase chain reaction (PCR), such as is described inthe handbook by Newton and Graham (PCR, Spektrum Akademischer Verlag,Heidelberg, 1994), can furthermore be used for the mutagenesis.

[0093] In addition, it may be advantageous for the production of L-aminoacids to enhance, in particular over-express, one or more enzymes of theparticular biosynthesis pathway, of glycolysis, of anaplerosis, of thecitric acid cycle, of the pentose phosphate cycle, of amino acid exportand optionally regulatory proteins, in addition to the rpsL gene.

[0094] Thus, for the preparation of L-lysine, in addition to enhancementof the rpsL gene, one or more genes chosen from the group consisting of

[0095] the dapA gene which codes for dihydrodipicolinate synthase (EP-B0 197 335),

[0096] the gap gene which codes for glyceraldehyde 3-phosphatedehydrogenase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086),

[0097] the tpi gene which codes for triose phosphate isomerase (Eikmanns(1992), Journal of Bacteriology 174:6076-6086),

[0098] the pgk gene which codes for 3-phosphoglycerate kinase (Eikmanns(1992), Journal of Bacteriology 174:6076-6086),

[0099] the zwf gene which codes for glucose 6-phosphate dehydrogenase(JP-A-09224661),

[0100] the pyc gene which codes for pyruvate carboxylase (DE-A-25 198 31609),

[0101] the mqo gene which codes for malate-quinone oxidoreductase(Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)),

[0102] the lysC gene which codes for a feed-back resistant aspartatekinase (Kalinowski et al. (1990), Molecular Microbiologie [sic] 5(5),1197-204 (1991)),

[0103] the lysE gene which codes for lysine export (DE-A-195 48 222),

[0104] the zwa1 gene which codes for the Zwal protein (DE: 19959328.0,DSM 13115), and

[0105] the rpoB gene which codes for the P-subunit of RNA polymerase B,shown in SEQ ID No. 5 and 6 can be enhanced, in particularover-expressed.

[0106] The term “attenuation” in this connection describes the reductionor elimination of the intracellular activity of one or more enzymes(proteins) in a microorganism which are coded by the corresponding DNA,for example by using a weak promoter or using a gene or allele whichcodes for a corresponding enzyme with a low activity or inactivates thecorresponding gene or enzyme (protein), and optionally combining thesemeasures.

[0107] It may furthermore be advantageous for the production of L-aminoacids, in addition to the enhancement of the rpsL gene, for one or moregenes chosen from the group consisting of:

[0108] the pck gene which codes for phosphoenol pyruvate carboxykinase(DE 199 50 409.1; DSM 13047),

[0109] the pgi gene which codes for glucose 6-phosphate isomerase (US09/396,478; DSM 12969),

[0110] the poxB gene which codes for pyruvate oxidase (DE: 1995 1975.7;DSM 13114),

[0111] the zwa2 gene which codes for the Zwa2 protein (DE: 19959327.2,DSM 13113) to be attenuated, in particular for the expression thereof tobe reduced.

[0112] In addition to enhancement of the rpsL gene it may furthermore beadvantageous for the production of amino acids to eliminate undesirableside reactions (Nakayama: “Breeding of Amino Acid ProducingMicro-organisms”, in: Overproduction of Microbial Products, Krumphanzl,Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).

[0113] The invention also provides the microorganisms prepared accordingto the invention, and these can be cultured continuously ordiscontinuously in the batch process (batch culture) or in the fed batch(feed process) or repeated fed batch process (repetitive feed process)for the purpose of production of amino acids. A summary of known culturemethods is described in the textbook by Chmiel (Bioprozesstechnik 1.Einführung in die Bioverfahrenstechnik [Bioprocess Technology 1.Introduction to Bioprocess Technology (Gustav Fischer Verlag, Stuttgart,1991)) or in the textbook by Storhas (Bioreaktoren und periphereEinrichtungen [Bioreactors and Peripheral Equipment] (Vieweg Verlag,Braunschweig/Wiesbaden, 1994)).

[0114] The culture medium to be used must meet the requirements of theparticular strains in a suitable manner. Descriptions of culture mediafor various microorganisms are contained in the handbook “Manual ofMethods for General Bacteriology” of the American Society forBacteriology (Washington D.C., USA, 1981).

[0115] Sugars and carbohydrates, such as e.g. glucose, sucrose, lactose,fructose, maltose, molasses, starch and cellulose, oils and fats, suchas e.g. soya oil, sunflower oil, groundnut oil and coconut fat, fattyacids, such as e.g. palmitic acid, stearic acid and linoleic acid,alcohols, such as e.g. glycerol and ethanol, and organic acids, such ase.g. acetic acid, can be used as the source of carbon. These substancecan be used individually or as a mixture.

[0116] Organic nitrogen-containing compounds, such as peptones, yeastextract, meat extract, malt extract, corn steep liquor, soya bean flourand urea, or inorganic compounds, such as ammonium sulfate, ammoniumchloride, ammonium phosphate, ammonium carbonate and ammonium nitrate,can be used as the source of nitrogen. The sources of nitrogen can beused individually or as a mixture.

[0117] Phosphoric acid, potassium dihydrogen phosphate or dipotassiumhydrogen phosphate or the corresponding sodium-containing salts can beused as the source of phosphorus. The culture medium must furthermorecomprise salts of metals, such as e. g. magnesium sulfate or ironsulfate, which are necessary for growth. Finally, essential growthsubstances, such as amino acids and vitamins, can be employed inaddition to the abovementioned substances. Suitable precursors canmoreover be added to the culture medium. The starting substancesmentioned can be added to the culture in the form of a single batch, orcan be fed in during the culture in a suitable manner.

[0118] Basic compounds, such as sodium hydroxide, potassium hydroxide,ammonia or aqueous ammonia, or acid compounds, such as phosphoric acidor sulfuric acid, can be employed in a suitable manner to control the pHof the culture. Antifoams, such as e.g. fatty acid polyglycol esters,can be employed to control the development of foam. Suitable substanceshaving a selective action, such as e.g. antibiotics, can be added to themedium to maintain the stability of plasmids. To maintain aerobicconditions, oxygen or oxygen-containing gas mixtures, such as e.g. air,are introduced into the culture. The temperature of the culture isusually 20° C. to 45° C., and preferably 25° C. to 40° C. Culturing iscontinued until a maximum of the desired product has formed. This targetis usually reached within 10 hours to 160 hours.

[0119] Methods for the determination of L-amino acids are known from theprior art. The analysis can thus be carried out, for example, asdescribed by Spackman et al. (Analytical Chemistry, 30, (1958), 1190) byion exchange chromatography with subsequent ninhydrin derivatization, orit can be carried out by reversed phase HPLC, for example as describedby Lindroth et al. (Analytical Chemistry (1979) 51: 1167-1174).

[0120] A pure culture of the Corynebacterium glutamicum strain DM1545was deposited on Jan. 16, 2001 at the Deutsche Sammlung fürMikrorganismen [sic] und Zellkulturen (DSMZ=German Collection ofMicroorganisms and Cell Cultures, Braunschweig, Germany) in accordancewith the Budapest Treaty as DSM 13992.

[0121] The process according to the invention is used for thefermentative preparation of amino acids, in particular L-lysine.

[0122] The present invention is explained in more detail in thefollowing with the aid of embodiment examples.

[0123] The isolation of plasmid DNA from Escherichia coli and alltechniques of restriction, Klenow and alkaline phosphatase treatmentwere carried out by the method of Sambrook et al. (Molecular Cloning. ALaboratory Manual (1989) Cold Spring Harbour [sic] Laboratory Press,Cold Spring Harbor, N.Y., USA). Methods for transformation ofEscherichia coli are also described in this handbook.

[0124] The composition of the usual nutrient media, such as LB or TYmedium, can also be found in the handbook by Sambrook et al. Havinggenerally described this invention, a further understanding can beobtained by reference to certain specific examples which are providedherein for purposes of illustration only, and are not intended to belimiting unless otherwise specified.

EXAMPLE 1

[0125] Preparation of a Genomic Cosmid Gene Library from Corynebacteriumglutamicum ATCC 13032

[0126] Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 isisolated as described by Tauch et al. (1995, Plasmid 33:168-179) andpartly cleaved with the restriction enzyme Sau3AI (Amersham Pharmacia,Freiburg, Germany, Product Description Sau3AI, Code no. 27-0913-02). TheDNA fragments are dephosphorylated with shrimp alkaline phosphatase(Roche Diagnostics GmbH, Mannheim, Germany, Product Description SAP,Code no. 1758250). The DNA of the cosmid vector SuperCos1 (Wahl et al.(1987) Proceedings of the National Academy of Sciences USA84:2160-2164), obtained from Stratagene (La Jolla, USA, ProductDescription SuperCosl Cosmid Vector Kit, Code no. 251301) is cleavedwith the restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany,Product Description XbaI, Code no. 27-0948-02) and likewisedephosphorylated with shrimp alkaline phosphatase.

[0127] The cosmid DNA is then cleaved with the restriction enzyme BamHI(Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Codeno. 27-0868-04). The cosmid DNA treated in this manner is mixed with thetreated ATCC13032 DNA and the batch is treated with T4 DNA ligase(Amersham Pharmacia, Freiburg, Germany, Product DescriptionT4-DNA-Ligase, Code no.27-0870-04). The ligation mixture is then packedin phages with the aid of Gigapack II XL Packing Extract (Stratagene, LaJolla, USA, Product Description Gigapack II XL Packing Extract, Code no.200217).

[0128] For infection of the E. coli strain NM554 (Raleigh et al. 1988,Nucleic Acid Research 16:1563-1575) the cells are taken up in 10 mMMgSO₄ and mixed with an aliquot of the phage suspension. The infectionand titering of the cosmid library are carried out as described bySambrook et al. (1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor), the cells being plated out on LB agar (Lennox, 1955,Virology, 1:190) with 100 mg/l ampicillin. After incubation overnight at37° C., recombinant individual clones are selected.

EXAMPLE 2

[0129] Isolation and Sequencing of the rpsL Gene

[0130] The cosmid DNA of an individual colony is isolated with theQiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany)in accordance with the manufacturer's instructions and partly cleavedwith the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg,Germany, Product Description Sau3AI, Product No. 27-0913-02). The DNAfragments are dephosphorylated with shrimp alkaline phosphatase (RocheDiagnostics GmbH, Mannheim, Germany, Product Description SAP, ProductNo. 1758250). After separation by gel electrophoresis, the cosmidfragments in the size range of 1500 to 2000 bp are isolated with theQiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany).

[0131] The DNA of the sequencing vector pZero-1, obtained fromInvitrogen (Groningen, Holland, Product Description Zero BackgroundCloning Kit, Product No. K2500-01), is cleaved with the restrictionenzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product DescriptionBamHI, Product No. 27-0868-04). The ligation of the cosmid fragments inthe sequencing vector pzero-l is carried out as described by Sambrook etal. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor),the DNA mixture being incubated overnight with T4 ligase (PharmaciaBiotech, Freiburg, Germany). This ligation mixture is thenelectroporated (Tauch et al. 1994, FEMS Microbiol Letters, 123:343-7)into the E. coli strain DH5αMCR (Grant, 1990, Proceedings of theNational Academy of Sciences U.S.A., 87:4645-4649) and plated out on LBagar (Lennox, 1955, Virology, 1:190) with 50 mg/l zeocin.

[0132] The plasmid preparation of the recombinant clones is carried outwith a Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany). Thesequencing is carried out by the dideoxy chain termination method ofSanger et al. (1977, Proceedings of the National Academy of SciencesU.S.A., 74:5463-5467) with modifications according to Zimmermann et al.(1990, Nucleic Acids Research, 18:1067). The “RR dRhodamin TerminatorCycle Sequencing Kit” from PE Applied Biosystems (Product No. 403044,Weiterstadt, Germany) is used. The separation by gel electrophoresis andanalysis of the sequencing reaction are carried out in a “RotiphoresisNF Acrylamide/Bisacrylamide” Gel (29:1) (Product No. A124.1, Roth,Karlsruhe, Germany) with the “ABI Prism 377” sequencer from PE AppliedBiosystems (Weiterstadt, Germany).

[0133] The raw sequence data obtained are then processed using theStaden program package (1986, Nucleic Acids Research, 14:217-231)version 97-0. The individual sequences of the pZerol derivatives areassembled to a continuous contig. The computer-assisted coding regionanalysis is prepared with the XNIP program (Staden, 1986, Nucleic AcidsResearch 14:217-231).

[0134] The resulting nucleotide sequence is shown in SEQ ID No. 1.Analysis of the nucleotide sequence shows an open reading frame of 383base pairs, which is called the rpsL gene. The rspL gene codes for aprotein of 127 amino acids. Obviously, numerous modifications andvariations on the present invention are possible in light of the aboveteachings. It is therefore to be understood that within the scope of theappended claims, the invention may be practiced otherwise than asspecifically described herein.

1 6 1 1775 DNA Corynebacterium glutamicum CDS (500)..(880) 1 cagctctacaagagtgtcta agtggcgggc attccatgct ttggaggagc gatcttcaaa 60 ttcctccaaagtgagttgac ctcgggaaac agctgcagaa agttcatcca cgacttggtt 120 tcggttaaggtcagtggcga gcttctttgc tggttcgttt ccttgaggaa cagtcatggg 180 aaccattctaacaagggatt tggtgttttc tgcggctagc tgataatgtg aacggctgag 240 tcccactcttgtagttggga attgacggca cctcgcactc aagcgcggta tcgcccctgg 300 ttttccgggacgcggtggcg catgtttgca tttgatgagg ttgtccgtga catgtttggt 360 cgggccccaaaaagagcccc cttttttgcg tgtctggaca ctttttcaaa tccttcgcca 420 tcgacaagctcagccttcgt gttcgtcccc cgggcgtcac gtcagcagtt aaagaacaac 480 tccgaaataaggatggttc atg cca act att cag cag ctg gtc cgt aag ggc 532 Met Pro ThrIle Gln Gln Leu Val Arg Lys Gly 1 5 10 cgc cac gat aag tcc gcc aag gtggct acc gcg gca ctg aag ggt tcc 580 Arg His Asp Lys Ser Ala Lys Val AlaThr Ala Ala Leu Lys Gly Ser 15 20 25 cct cag cgt cgt ggc gta tgc acc cgtgtg tac acc acc acc cct aag 628 Pro Gln Arg Arg Gly Val Cys Thr Arg ValTyr Thr Thr Thr Pro Lys 30 35 40 aag cct aac tct gct ctt cgt aag gtc gctcgt gtg cgc ctt acc tcc 676 Lys Pro Asn Ser Ala Leu Arg Lys Val Ala ArgVal Arg Leu Thr Ser 45 50 55 ggc atc gag gtt tcc gct tac atc cct ggt gagggc cac aac ctg cag 724 Gly Ile Glu Val Ser Ala Tyr Ile Pro Gly Glu GlyHis Asn Leu Gln 60 65 70 75 gag cac tcc atg gtg ctc gtt cgc ggt ggt cgtgtt aag gac ctc cca 772 Glu His Ser Met Val Leu Val Arg Gly Gly Arg ValLys Asp Leu Pro 80 85 90 ggt gtc cgt tac aag atc gtc cgt ggc gca ctg gatacc cag ggt gtt 820 Gly Val Arg Tyr Lys Ile Val Arg Gly Ala Leu Asp ThrGln Gly Val 95 100 105 aag gac cgc aag cag gct cgt tcc ccg cta cgg cgcgaa gag ggg ata 868 Lys Asp Arg Lys Gln Ala Arg Ser Pro Leu Arg Arg GluGlu Gly Ile 110 115 120 att aaa aat gcg taaatcagca gctcctaagc gtccagtagttcaggaccct 920 Ile Lys Asn Ala 125 gtatacaagt ccgagctcgt tacccagctcgtaaacaaga tcctcatcgg tggcaagaag 980 tccaccgcag agcgcatcgt ctacggtgcactcgagatct gccgtgagaa gaccggcacc 1040 gatccagtag gaaccctcga gaaggctctcggcaacgtgc gtccagacct cgaagttcgt 1100 tcccgccgtg ttggtggcgc tacctaccaggtgccagtgg atgttcgccc agagcgcgca 1160 aacaccctcg cactgcgttg gttggtaaccttcacccgtc agcgtcgtga gaacaccatg 1220 atcgagcgtc ttgcaaacga acttctggatgcagccaacg gccttggcgc ttccgtgaag 1280 cgtcgcgaag acacccacaa gatggcagaggccaaccgcg ccttcgctca ctaccgctgg 1340 tagtactgcc aagacatgaa agcccaatcacctttaagat caacgcctgc cggcgccctt 1400 cacatttgaa taagctggca gcctgcgtttcttcaaggcg actgggcttt tagtctcatt 1460 aatgcagttc accgctgtaa gatagctaaatagaaacact gtttcggcag tgtgttacta 1520 aaaaatccat gtcacttgcc tcgagcgtgctgcttgaatc gcaagttagt ggcaaaatgt 1580 aacaagagaa ttatccgtag gtgacaaactttttaatact tgggtatctg tcatggatac 1640 cccggtaata aataagtgaa ttaccgtaaccaacaagttg gggtaccact gtggcacaag 1700 aagtgcttaa ggatctaaac aaggtccgcaacatcggcat catggcgcac atcgatgctg 1760 gtaagaccac gacca 1775 2 127 PRTCorynebacterium glutamicum 2 Met Pro Thr Ile Gln Gln Leu Val Arg Lys GlyArg His Asp Lys Ser 1 5 10 15 Ala Lys Val Ala Thr Ala Ala Leu Lys GlySer Pro Gln Arg Arg Gly 20 25 30 Val Cys Thr Arg Val Tyr Thr Thr Thr ProLys Lys Pro Asn Ser Ala 35 40 45 Leu Arg Lys Val Ala Arg Val Arg Leu ThrSer Gly Ile Glu Val Ser 50 55 60 Ala Tyr Ile Pro Gly Glu Gly His Asn LeuGln Glu His Ser Met Val 65 70 75 80 Leu Val Arg Gly Gly Arg Val Lys AspLeu Pro Gly Val Arg Tyr Lys 85 90 95 Ile Val Arg Gly Ala Leu Asp Thr GlnGly Val Lys Asp Arg Lys Gln 100 105 110 Ala Arg Ser Pro Leu Arg Arg GluGlu Gly Ile Ile Lys Asn Ala 115 120 125 3 1775 DNA Corynebacteriumglutamicum CDS (500)..(880) 3 cagctctaca agagtgtcta agtggcgggcattccatgct ttggaggagc gatcttcaaa 60 ttcctccaaa gtgagttgac ctcgggaaacagctgcagaa agttcatcca cgacttggtt 120 tcggttaagg tcagtggcga gcttctttgctggttcgttt ccttgaggaa cagtcatggg 180 aaccattcta acaagggatt tggtgttttctgcggctagc tgataatgtg aacggctgag 240 tcccactctt gtagttggga attgacggcacctcgcactc aagcgcggta tcgcccctgg 300 ttttccggga cgcggtggcg catgtttgcatttgatgagg ttgtccgtga catgtttggt 360 cgggccccaa aaagagcccc cttttttgcgtgtctggaca ctttttcaaa tccttcgcca 420 tcgacaagct cagccttcgt gttcgtcccccgggcgtcac gtcagcagtt aaagaacaac 480 tccgaaataa ggatggttc atg cca actatt cag cag ctg gtc cgt aag ggc 532 Met Pro Thr Ile Gln Gln Leu Val ArgLys Gly 1 5 10 cgc cac gat aag tcc gcc aag gtg gct acc gcg gca ctg aagggt tcc 580 Arg His Asp Lys Ser Ala Lys Val Ala Thr Ala Ala Leu Lys GlySer 15 20 25 cct cag cgt cgt ggc gta tgc acc cgt gtg tac acc acc acc cctagg 628 Pro Gln Arg Arg Gly Val Cys Thr Arg Val Tyr Thr Thr Thr Pro Arg30 35 40 aag cct aac tct gct ctt cgt aag gtc gct cgt gtg cgc ctt acc tcc676 Lys Pro Asn Ser Ala Leu Arg Lys Val Ala Arg Val Arg Leu Thr Ser 4550 55 ggc atc gag gtt tcc gct tac atc cct ggt gag ggc cac aac ctg cag724 Gly Ile Glu Val Ser Ala Tyr Ile Pro Gly Glu Gly His Asn Leu Gln 6065 70 75 gag cac tcc atg gtg ctc gtt cgc ggt ggt cgt gtt aag gac ctc cca772 Glu His Ser Met Val Leu Val Arg Gly Gly Arg Val Lys Asp Leu Pro 8085 90 ggt gtc cgt tac aag atc gtc cgt ggc gca ctg gat acc cag ggt gtt820 Gly Val Arg Tyr Lys Ile Val Arg Gly Ala Leu Asp Thr Gln Gly Val 95100 105 aag gac cgc aag cag gct cgt tcc ccg cta cgg cgc gaa gag ggg ata868 Lys Asp Arg Lys Gln Ala Arg Ser Pro Leu Arg Arg Glu Glu Gly Ile 110115 120 att aaa aat gcg taaatcagca gctcctaagc gtccagtagt tcaggaccct 920Ile Lys Asn Ala 125 gtatacaagt ccgagctcgt tacccagctc gtaaacaagatcctcatcgg tggcaagaag 980 tccaccgcag agcgcatcgt ctacggtgca ctcgagatctgccgtgagaa gaccggcacc 1040 gatccagtag gaaccctcga gaaggctctc ggcaacgtgcgtccagacct cgaagttcgt 1100 tcccgccgtg ttggtggcgc tacctaccag gtgccagtggatgttcgccc agagcgcgca 1160 aacaccctcg cactgcgttg gttggtaacc ttcacccgtcagcgtcgtga gaacaccatg 1220 atcgagcgtc ttgcaaacga acttctggat gcagccaacggccttggcgc ttccgtgaag 1280 cgtcgcgaag acacccacaa gatggcagag gccaaccgcgccttcgctca ctaccgctgg 1340 tagtactgcc aagacatgaa agcccaatca cctttaagatcaacgcctgc cggcgccctt 1400 cacatttgaa taagctggca gcctgcgttt cttcaaggcgactgggcttt tagtctcatt 1460 aatgcagttc accgctgtaa gatagctaaa tagaaacactgtttcggcag tgtgttacta 1520 aaaaatccat gtcacttgcc tcgagcgtgc tgcttgaatcgcaagttagt ggcaaaatgt 1580 aacaagagaa ttatccgtag gtgacaaact ttttaatacttgggtatctg tcatggatac 1640 cccggtaata aataagtgaa ttaccgtaac caacaagttggggtaccact gtggcacaag 1700 aagtgcttaa ggatctaaac aaggtccgca acatcggcatcatggcgcac atcgatgctg 1760 gtaagaccac gacca 1775 4 127 PRTCorynebacterium glutamicum 4 Met Pro Thr Ile Gln Gln Leu Val Arg Lys GlyArg His Asp Lys Ser 1 5 10 15 Ala Lys Val Ala Thr Ala Ala Leu Lys GlySer Pro Gln Arg Arg Gly 20 25 30 Val Cys Thr Arg Val Tyr Thr Thr Thr ProArg Lys Pro Asn Ser Ala 35 40 45 Leu Arg Lys Val Ala Arg Val Arg Leu ThrSer Gly Ile Glu Val Ser 50 55 60 Ala Tyr Ile Pro Gly Glu Gly His Asn LeuGln Glu His Ser Met Val 65 70 75 80 Leu Val Arg Gly Gly Arg Val Lys AspLeu Pro Gly Val Arg Tyr Lys 85 90 95 Ile Val Arg Gly Ala Leu Asp Thr GlnGly Val Lys Asp Arg Lys Gln 100 105 110 Ala Arg Ser Pro Leu Arg Arg GluGlu Gly Ile Ile Lys Asn Ala 115 120 125 5 5096 DNA Corynebacteriumglutamicum CDS (702)..(4196) 5 acaatgtgac tcgtgatttt tgggtggatcagcgtaccgg tttggttgtc gatctagctg 60 aaaatattga tgatttttac ggcgaccgcagcggccagaa gtacgaacag aaattgcttt 120 tcgacgcctc cctcgacgat gcagctgtctctaagctggt tgcacaggcc gaaagcatcc 180 ctgatggaga tgtgagcaaa atcgcaaataccgtaggtat tgtgatcggt gcggtattgg 240 ctctcgtggg cctggccggg tgttttggggcgtttgggaa gaaacgtcga gaagcttaac 300 ctgctgttca aatagatttt ccctgtttcgaattgcggaa accccgggtt tgtttgctag 360 ggtgcctcgt agaaggggtc aagaagatttctgggaaacg cgcccgtgcg gttggttgct 420 aatagcacgc ggagcaccag atgaaaaatctcccctttac tttcgcgcgc gattggtata 480 ctctgagtcg ttgcgttgga attcgtgactctttttcgtt cctgtagcgc caagaccttg 540 atcaaggtgg tttaaaaaaa ccgatttgacaaggtcattc agtgctatct ggagtcgttc 600 agggggatcg ggttcctcag cagaccaattgctcaaaaat accagcggtg ttgatctgca 660 cttaatggcc ttgaccagcc aggtgcaattacccgcgtga g gtg ctg gaa gga ccc 716 Val Leu Glu Gly Pro 1 5 atc ttg gcagtc tcc cgc cag acc aag tca gtc gtc gat att ccc ggt 764 Ile Leu Ala ValSer Arg Gln Thr Lys Ser Val Val Asp Ile Pro Gly 10 15 20 gca ccg cag cgttat tct ttc gcg aag gtg tcc gca ccc att gag gtg 812 Ala Pro Gln Arg TyrSer Phe Ala Lys Val Ser Ala Pro Ile Glu Val 25 30 35 ccc ggg cta cta gatctt caa ctg gat tct tac tcc tgg ctg att ggt 860 Pro Gly Leu Leu Asp LeuGln Leu Asp Ser Tyr Ser Trp Leu Ile Gly 40 45 50 acg cct gag tgg cgt gctcgt cag aag gaa gaa ttc ggc gag gga gcc 908 Thr Pro Glu Trp Arg Ala ArgGln Lys Glu Glu Phe Gly Glu Gly Ala 55 60 65 cgc gta acc agc ggc ctt gagaac att ctc gag gag ctc tcc cca atc 956 Arg Val Thr Ser Gly Leu Glu AsnIle Leu Glu Glu Leu Ser Pro Ile 70 75 80 85 cag gat tac tct gga aac atgtcc ctg agc ctt tcg gag cca cgc ttc 1004 Gln Asp Tyr Ser Gly Asn Met SerLeu Ser Leu Ser Glu Pro Arg Phe 90 95 100 gaa gac gtc aag aac acc attgac gag gcg aaa gaa aag gac atc aac 1052 Glu Asp Val Lys Asn Thr Ile AspGlu Ala Lys Glu Lys Asp Ile Asn 105 110 115 tac gcg gcg cca ctg tat gtgacc gcg gag ttc gtc aac aac acc acc 1100 Tyr Ala Ala Pro Leu Tyr Val ThrAla Glu Phe Val Asn Asn Thr Thr 120 125 130 ggt gaa atc aag tct cag actgtc ttc atc ggc gat ttc cca atg atg 1148 Gly Glu Ile Lys Ser Gln Thr ValPhe Ile Gly Asp Phe Pro Met Met 135 140 145 acg gac aag gga acg ttc atcatc aac gga acc gaa cgc gtt gtg gtc 1196 Thr Asp Lys Gly Thr Phe Ile IleAsn Gly Thr Glu Arg Val Val Val 150 155 160 165 agc cag ctc gtc cgc tccccg ggc gtg tac ttt gac cag acc atc gat 1244 Ser Gln Leu Val Arg Ser ProGly Val Tyr Phe Asp Gln Thr Ile Asp 170 175 180 aag tca act gag cgt ccactg cac gcc gtg aag gtt att cct tcc cgt 1292 Lys Ser Thr Glu Arg Pro LeuHis Ala Val Lys Val Ile Pro Ser Arg 185 190 195 ggt gct tgg ctt gag tttgac gtc gat aag cgc gat tcg gtt ggt gtt 1340 Gly Ala Trp Leu Glu Phe AspVal Asp Lys Arg Asp Ser Val Gly Val 200 205 210 cgt att gac cgc aag cgtcgc cag cca gtc acc gta ctg ctg aag gct 1388 Arg Ile Asp Arg Lys Arg ArgGln Pro Val Thr Val Leu Leu Lys Ala 215 220 225 ctt ggc tgg acc act gagcag atc acc gag cgt ttc ggt ttc tct gaa 1436 Leu Gly Trp Thr Thr Glu GlnIle Thr Glu Arg Phe Gly Phe Ser Glu 230 235 240 245 atc atg atg tcc accctc gag tcc gat ggt gta gca aac acc gat gag 1484 Ile Met Met Ser Thr LeuGlu Ser Asp Gly Val Ala Asn Thr Asp Glu 250 255 260 gca ttg ctg gag atctac cgc aag cag cgt cca ggc gag cag cct acc 1532 Ala Leu Leu Glu Ile TyrArg Lys Gln Arg Pro Gly Glu Gln Pro Thr 265 270 275 cgc gac ctt gcg cagtcc ctc ctg gac aac agc ttc ttc cgt gca aag 1580 Arg Asp Leu Ala Gln SerLeu Leu Asp Asn Ser Phe Phe Arg Ala Lys 280 285 290 cgc tac gac ctg gctcgc gtt ggt cgt tac aag atc aac cgc aag ctc 1628 Arg Tyr Asp Leu Ala ArgVal Gly Arg Tyr Lys Ile Asn Arg Lys Leu 295 300 305 ggc ctt ggt ggc gaccac gat ggt ttg atg act ctt act gaa gag gac 1676 Gly Leu Gly Gly Asp HisAsp Gly Leu Met Thr Leu Thr Glu Glu Asp 310 315 320 325 atc gca acc accatc gag tac ctg gtg cgt ctg cac gca ggt gag cgc 1724 Ile Ala Thr Thr IleGlu Tyr Leu Val Arg Leu His Ala Gly Glu Arg 330 335 340 gtc atg act tctcca aat ggt gaa gag atc cca gtc gag acc gat gac 1772 Val Met Thr Ser ProAsn Gly Glu Glu Ile Pro Val Glu Thr Asp Asp 345 350 355 atc gac cac tttggt aac cgt cgt ctg cgt acc gtt ggc gaa ctg atc 1820 Ile Asp His Phe GlyAsn Arg Arg Leu Arg Thr Val Gly Glu Leu Ile 360 365 370 cag aac cag gtccgt gtc ggc ctg tcc cgc atg gag cgc gtt gtt cgt 1868 Gln Asn Gln Val ArgVal Gly Leu Ser Arg Met Glu Arg Val Val Arg 375 380 385 gag cgt atg accacc cag gat gcg gag tcc att act cct act tcc ttg 1916 Glu Arg Met Thr ThrGln Asp Ala Glu Ser Ile Thr Pro Thr Ser Leu 390 395 400 405 atc aac gttcgt cct gtc tct gca gct atc cgt gag ttc ttc gga act 1964 Ile Asn Val ArgPro Val Ser Ala Ala Ile Arg Glu Phe Phe Gly Thr 410 415 420 tcc cag ctgtct cag ttc atg gtc cag aac aac tcc ctg tct ggt ttg 2012 Ser Gln Leu SerGln Phe Met Val Gln Asn Asn Ser Leu Ser Gly Leu 425 430 435 act cac aagcgt cgt ctg tcg gct ctg ggc ccg ggt ggt ctg tcc cgt 2060 Thr His Lys ArgArg Leu Ser Ala Leu Gly Pro Gly Gly Leu Ser Arg 440 445 450 gag cgc gccggc atc gag gtt cga gac gtt cac cca tct cac tac ggc 2108 Glu Arg Ala GlyIle Glu Val Arg Asp Val His Pro Ser His Tyr Gly 455 460 465 cgt atg tgccca att gag act ccg gaa ggt cca aac att ggc ctg atc 2156 Arg Met Cys ProIle Glu Thr Pro Glu Gly Pro Asn Ile Gly Leu Ile 470 475 480 485 ggt tccttg gct tcc tat gct cga gtg aac cca ttc ggt ttc att gag 2204 Gly Ser LeuAla Ser Tyr Ala Arg Val Asn Pro Phe Gly Phe Ile Glu 490 495 500 acc ccatac cgt cgc atc atc gac ggc aag ctg acc gac cag att gac 2252 Thr Pro TyrArg Arg Ile Ile Asp Gly Lys Leu Thr Asp Gln Ile Asp 505 510 515 tac cttacc gct gat gag gaa gac cgc ttc gtt gtt gcg cag gca aac 2300 Tyr Leu ThrAla Asp Glu Glu Asp Arg Phe Val Val Ala Gln Ala Asn 520 525 530 acg cactac gac gaa gag ggc aac atc acc gat gag acc gtc act gtt 2348 Thr His TyrAsp Glu Glu Gly Asn Ile Thr Asp Glu Thr Val Thr Val 535 540 545 cgt ctgaag gac ggc gac atc gcc atg gtt ggc cgc aac gcg gtt gat 2396 Arg Leu LysAsp Gly Asp Ile Ala Met Val Gly Arg Asn Ala Val Asp 550 555 560 565 tacatg gac gtt tcc cct cgt cag atg gtt tct gtt ggt acc gcg atg 2444 Tyr MetAsp Val Ser Pro Arg Gln Met Val Ser Val Gly Thr Ala Met 570 575 580 attcca ttc ctg gag cac gac gat gct aac cgt gca ctg atg ggc gcg 2492 Ile ProPhe Leu Glu His Asp Asp Ala Asn Arg Ala Leu Met Gly Ala 585 590 595 aacatg cag aag cag gct gtg cca ctg att cgt gcc gag gct cct ttc 2540 Asn MetGln Lys Gln Ala Val Pro Leu Ile Arg Ala Glu Ala Pro Phe 600 605 610 gtgggc acc ggt atg gag cag cgc gca gca tac gac gcc ggc gac ctg 2588 Val GlyThr Gly Met Glu Gln Arg Ala Ala Tyr Asp Ala Gly Asp Leu 615 620 625 gttatt acc cca gtc gca ggt gtg gtg gaa aac gtt tca gct gac ttc 2636 Val IleThr Pro Val Ala Gly Val Val Glu Asn Val Ser Ala Asp Phe 630 635 640 645atc acc atc atg gct gat gac ggc aag cgc gaa acc tac ctg ctg cgt 2684 IleThr Ile Met Ala Asp Asp Gly Lys Arg Glu Thr Tyr Leu Leu Arg 650 655 660aag ttc cag cgc acc aac cag ggc acc agc tac aac cag aag cct ttg 2732 LysPhe Gln Arg Thr Asn Gln Gly Thr Ser Tyr Asn Gln Lys Pro Leu 665 670 675gtt aac ttg ggc gag cgc gtt gaa gct ggc cag gtt att gct gat ggt 2780 ValAsn Leu Gly Glu Arg Val Glu Ala Gly Gln Val Ile Ala Asp Gly 680 685 690cca ggt acc ttc aat ggt gaa atg tcc ctt ggc cgt aac ctt ctg gtt 2828 ProGly Thr Phe Asn Gly Glu Met Ser Leu Gly Arg Asn Leu Leu Val 695 700 705gcg ttc atg cct tgg gaa ggc cac aac tac gag gat gcg atc atc ctc 2876 AlaPhe Met Pro Trp Glu Gly His Asn Tyr Glu Asp Ala Ile Ile Leu 710 715 720725 aac cag aac atc gtt gag cag gac atc ttg acc tcg atc cac atc gag 2924Asn Gln Asn Ile Val Glu Gln Asp Ile Leu Thr Ser Ile His Ile Glu 730 735740 gag cac gag atc gat gcc cgc gac act aag ctt ggc gcc gaa gaa atc 2972Glu His Glu Ile Asp Ala Arg Asp Thr Lys Leu Gly Ala Glu Glu Ile 745 750755 acc cgc gac atc cct aat gtg tct gaa gaa gtc ctc aag gac ctc gac 3020Thr Arg Asp Ile Pro Asn Val Ser Glu Glu Val Leu Lys Asp Leu Asp 760 765770 gac cgc ggt att gtc cgc atc ggt gct gat gtt cgt gac ggc gac atc 3068Asp Arg Gly Ile Val Arg Ile Gly Ala Asp Val Arg Asp Gly Asp Ile 775 780785 ctg gtc ggt aag gtc acc cct aag ggc gag acc gag ctc acc ccg gaa 3116Leu Val Gly Lys Val Thr Pro Lys Gly Glu Thr Glu Leu Thr Pro Glu 790 795800 805 gag cgc ttg ctg cgc gca atc ttc ggt gag aag gcc cgc gaa gtt gat3164 Glu Arg Leu Leu Arg Ala Ile Phe Gly Glu Lys Ala Arg Glu Val Asp 810815 820 acc tcc atg aag gtg cct cac ggt gag acc ggc aag gtc atc ggc gtg3212 Thr Ser Met Lys Val Pro His Gly Glu Thr Gly Lys Val Ile Gly Val 825830 835 cgt cac ttc tcc cgc gag gac gac gac gat ctg gct cct ggc gtc aac3260 Arg His Phe Ser Arg Glu Asp Asp Asp Asp Leu Ala Pro Gly Val Asn 840845 850 gag atg atc cgt atc tac gtt gct cag aag cgt aag atc cag gac ggc3308 Glu Met Ile Arg Ile Tyr Val Ala Gln Lys Arg Lys Ile Gln Asp Gly 855860 865 gat aag ctc gct ggc cgc cac ggt aac aag ggt gtt gtc ggt aaa att3356 Asp Lys Leu Ala Gly Arg His Gly Asn Lys Gly Val Val Gly Lys Ile 870875 880 885 ttg cct cag gaa gat atg cca ttc ctt cca gac ggc act cct gttgac 3404 Leu Pro Gln Glu Asp Met Pro Phe Leu Pro Asp Gly Thr Pro Val Asp890 895 900 atc atc ttg aac acc cac ggt gtt cca cgt cgt atg aac att ggtcag 3452 Ile Ile Leu Asn Thr His Gly Val Pro Arg Arg Met Asn Ile Gly Gln905 910 915 gtt ctt gag acc cac ctt ggc tgg ctg gca tct gct ggt tgg tccgtg 3500 Val Leu Glu Thr His Leu Gly Trp Leu Ala Ser Ala Gly Trp Ser Val920 925 930 gat cct gaa gat cct gag aac gct gag ctc gtc aag act ctg cctgca 3548 Asp Pro Glu Asp Pro Glu Asn Ala Glu Leu Val Lys Thr Leu Pro Ala935 940 945 gac ctc ctc gag gtt cct gct ggt tcc ttg act gca act cct gtgttc 3596 Asp Leu Leu Glu Val Pro Ala Gly Ser Leu Thr Ala Thr Pro Val Phe950 955 960 965 gac ggt gcg tca aac gaa gag ctc gca ggc ctg ctc gct aattca cgt 3644 Asp Gly Ala Ser Asn Glu Glu Leu Ala Gly Leu Leu Ala Asn SerArg 970 975 980 cca aac cgc gac ggc gac gtc atg gtt aac gcg gat ggt aaagca acg 3692 Pro Asn Arg Asp Gly Asp Val Met Val Asn Ala Asp Gly Lys AlaThr 985 990 995 ctt atc gac ggt cgc tcc ggt gag cct tac ccg tac ccg gtttcc 3737 Leu Ile Asp Gly Arg Ser Gly Glu Pro Tyr Pro Tyr Pro Val Ser1000 1005 1010 atc ggc tac atg tac atg ctg aag ctg cac cac ctc gtt gacgag 3782 Ile Gly Tyr Met Tyr Met Leu Lys Leu His His Leu Val Asp Glu1015 1020 1025 aag atc cac gca cgt tcc act ggt cct tac tcc atg att acccag 3827 Lys Ile His Ala Arg Ser Thr Gly Pro Tyr Ser Met Ile Thr Gln1030 1035 1040 cag cca ctg ggt ggt aaa gca cag ttc ggt gga cag cgt ttcggc 3872 Gln Pro Leu Gly Gly Lys Ala Gln Phe Gly Gly Gln Arg Phe Gly1045 1050 1055 gaa atg gag gtg tgg gca atg cag gca tac ggc gct gcc tacaca 3917 Glu Met Glu Val Trp Ala Met Gln Ala Tyr Gly Ala Ala Tyr Thr1060 1065 1070 ctt cag gag ctg ctg acc atc aag tct gat gac gtg gtt ggccgt 3962 Leu Gln Glu Leu Leu Thr Ile Lys Ser Asp Asp Val Val Gly Arg1075 1080 1085 gtc aag gtc tac gaa gca att gtg aag ggc gag aac atc ccggat 4007 Val Lys Val Tyr Glu Ala Ile Val Lys Gly Glu Asn Ile Pro Asp1090 1095 1100 cca ggt att cct gag tcc ttc aag gtt ctc ctc aag gag ctccag 4052 Pro Gly Ile Pro Glu Ser Phe Lys Val Leu Leu Lys Glu Leu Gln1105 1110 1115 tcc ttg tgc ctg aac gtg gag gtt ctc tcc gca gac ggc actcca 4097 Ser Leu Cys Leu Asn Val Glu Val Leu Ser Ala Asp Gly Thr Pro1120 1125 1130 atg gag ctc gcg ggt gac gac gac gac ttc gat cag gca ggcgcc 4142 Met Glu Leu Ala Gly Asp Asp Asp Asp Phe Asp Gln Ala Gly Ala1135 1140 1145 tca ctt ggc atc aac ctg tcc cgt gac gag cgt tcc gac gccgac 4187 Ser Leu Gly Ile Asn Leu Ser Arg Asp Glu Arg Ser Asp Ala Asp1150 1155 1160 acc gca tag cagatcagaa aacaaccgct agaaatcaag ccatacatcc4236 Thr Ala cccggacatt gaagagatgt tctgggggga aagggagttt tacgtgctcgacgtaaacgt 4296 cttcgatgag ctccgcatcg gcctggccac cgccgacgac atccgccgttggtccaaggg 4356 tgaggtcaag aagccggaga ccatcaacta ccgaaccctc aagcctgagaaggacggtct 4416 gttctgcgag cgtatcttcg gtccaactcg cgactgggag tgcgcctgcggtaagtacaa 4476 gcgtgtccgc tacaagggca tcatctgtga acgctgtggc gttgaggtcaccaagtccaa 4536 ggtgcgccgt gagcgcatgg gacacattga gctcgctgca ccagtaacccacatttggta 4596 cttcaagggc gttccatcac gcctcggcta ccttttggac cttgctccaaaggacctgga 4656 cctcatcatc tacttcggtg cgaacatcat caccagcgtg gacgaagaggctcgccacag 4716 cgaccagacc actcttgagg cagaaatgct tctggagaag aaggacgttgaggcagacgc 4776 agagtctgac attgctgagc gtgctgaaaa gctcgaagag gatcttgctgaacttgaggc 4836 agctggcgct aaggccgacg ctcgccgcaa ggttcaggct gctgccgataaggaaatgca 4896 gcacatccgt gagcgtgcac agcgcgaaat cgatcgtctc gatgaggtctggcagacctt 4956 catcaagctt gctccaaagc agatgatccg cgatgagaag ctctacgatgaactgatcga 5016 ccgctacgag gattacttca ccggtggtat gggtgcagag tccattgaggctttgatcca 5076 gaacttcgac cttgatgctg 5096 6 1164 PRT Corynebacteriumglutamicum 6 Val Leu Glu Gly Pro Ile Leu Ala Val Ser Arg Gln Thr Lys SerVal 1 5 10 15 Val Asp Ile Pro Gly Ala Pro Gln Arg Tyr Ser Phe Ala LysVal Ser 20 25 30 Ala Pro Ile Glu Val Pro Gly Leu Leu Asp Leu Gln Leu AspSer Tyr 35 40 45 Ser Trp Leu Ile Gly Thr Pro Glu Trp Arg Ala Arg Gln LysGlu Glu 50 55 60 Phe Gly Glu Gly Ala Arg Val Thr Ser Gly Leu Glu Asn IleLeu Glu 65 70 75 80 Glu Leu Ser Pro Ile Gln Asp Tyr Ser Gly Asn Met SerLeu Ser Leu 85 90 95 Ser Glu Pro Arg Phe Glu Asp Val Lys Asn Thr Ile AspGlu Ala Lys 100 105 110 Glu Lys Asp Ile Asn Tyr Ala Ala Pro Leu Tyr ValThr Ala Glu Phe 115 120 125 Val Asn Asn Thr Thr Gly Glu Ile Lys Ser GlnThr Val Phe Ile Gly 130 135 140 Asp Phe Pro Met Met Thr Asp Lys Gly ThrPhe Ile Ile Asn Gly Thr 145 150 155 160 Glu Arg Val Val Val Ser Gln LeuVal Arg Ser Pro Gly Val Tyr Phe 165 170 175 Asp Gln Thr Ile Asp Lys SerThr Glu Arg Pro Leu His Ala Val Lys 180 185 190 Val Ile Pro Ser Arg GlyAla Trp Leu Glu Phe Asp Val Asp Lys Arg 195 200 205 Asp Ser Val Gly ValArg Ile Asp Arg Lys Arg Arg Gln Pro Val Thr 210 215 220 Val Leu Leu LysAla Leu Gly Trp Thr Thr Glu Gln Ile Thr Glu Arg 225 230 235 240 Phe GlyPhe Ser Glu Ile Met Met Ser Thr Leu Glu Ser Asp Gly Val 245 250 255 AlaAsn Thr Asp Glu Ala Leu Leu Glu Ile Tyr Arg Lys Gln Arg Pro 260 265 270Gly Glu Gln Pro Thr Arg Asp Leu Ala Gln Ser Leu Leu Asp Asn Ser 275 280285 Phe Phe Arg Ala Lys Arg Tyr Asp Leu Ala Arg Val Gly Arg Tyr Lys 290295 300 Ile Asn Arg Lys Leu Gly Leu Gly Gly Asp His Asp Gly Leu Met Thr305 310 315 320 Leu Thr Glu Glu Asp Ile Ala Thr Thr Ile Glu Tyr Leu ValArg Leu 325 330 335 His Ala Gly Glu Arg Val Met Thr Ser Pro Asn Gly GluGlu Ile Pro 340 345 350 Val Glu Thr Asp Asp Ile Asp His Phe Gly Asn ArgArg Leu Arg Thr 355 360 365 Val Gly Glu Leu Ile Gln Asn Gln Val Arg ValGly Leu Ser Arg Met 370 375 380 Glu Arg Val Val Arg Glu Arg Met Thr ThrGln Asp Ala Glu Ser Ile 385 390 395 400 Thr Pro Thr Ser Leu Ile Asn ValArg Pro Val Ser Ala Ala Ile Arg 405 410 415 Glu Phe Phe Gly Thr Ser GlnLeu Ser Gln Phe Met Val Gln Asn Asn 420 425 430 Ser Leu Ser Gly Leu ThrHis Lys Arg Arg Leu Ser Ala Leu Gly Pro 435 440 445 Gly Gly Leu Ser ArgGlu Arg Ala Gly Ile Glu Val Arg Asp Val His 450 455 460 Pro Ser His TyrGly Arg Met Cys Pro Ile Glu Thr Pro Glu Gly Pro 465 470 475 480 Asn IleGly Leu Ile Gly Ser Leu Ala Ser Tyr Ala Arg Val Asn Pro 485 490 495 PheGly Phe Ile Glu Thr Pro Tyr Arg Arg Ile Ile Asp Gly Lys Leu 500 505 510Thr Asp Gln Ile Asp Tyr Leu Thr Ala Asp Glu Glu Asp Arg Phe Val 515 520525 Val Ala Gln Ala Asn Thr His Tyr Asp Glu Glu Gly Asn Ile Thr Asp 530535 540 Glu Thr Val Thr Val Arg Leu Lys Asp Gly Asp Ile Ala Met Val Gly545 550 555 560 Arg Asn Ala Val Asp Tyr Met Asp Val Ser Pro Arg Gln MetVal Ser 565 570 575 Val Gly Thr Ala Met Ile Pro Phe Leu Glu His Asp AspAla Asn Arg 580 585 590 Ala Leu Met Gly Ala Asn Met Gln Lys Gln Ala ValPro Leu Ile Arg 595 600 605 Ala Glu Ala Pro Phe Val Gly Thr Gly Met GluGln Arg Ala Ala Tyr 610 615 620 Asp Ala Gly Asp Leu Val Ile Thr Pro ValAla Gly Val Val Glu Asn 625 630 635 640 Val Ser Ala Asp Phe Ile Thr IleMet Ala Asp Asp Gly Lys Arg Glu 645 650 655 Thr Tyr Leu Leu Arg Lys PheGln Arg Thr Asn Gln Gly Thr Ser Tyr 660 665 670 Asn Gln Lys Pro Leu ValAsn Leu Gly Glu Arg Val Glu Ala Gly Gln 675 680 685 Val Ile Ala Asp GlyPro Gly Thr Phe Asn Gly Glu Met Ser Leu Gly 690 695 700 Arg Asn Leu LeuVal Ala Phe Met Pro Trp Glu Gly His Asn Tyr Glu 705 710 715 720 Asp AlaIle Ile Leu Asn Gln Asn Ile Val Glu Gln Asp Ile Leu Thr 725 730 735 SerIle His Ile Glu Glu His Glu Ile Asp Ala Arg Asp Thr Lys Leu 740 745 750Gly Ala Glu Glu Ile Thr Arg Asp Ile Pro Asn Val Ser Glu Glu Val 755 760765 Leu Lys Asp Leu Asp Asp Arg Gly Ile Val Arg Ile Gly Ala Asp Val 770775 780 Arg Asp Gly Asp Ile Leu Val Gly Lys Val Thr Pro Lys Gly Glu Thr785 790 795 800 Glu Leu Thr Pro Glu Glu Arg Leu Leu Arg Ala Ile Phe GlyGlu Lys 805 810 815 Ala Arg Glu Val Asp Thr Ser Met Lys Val Pro His GlyGlu Thr Gly 820 825 830 Lys Val Ile Gly Val Arg His Phe Ser Arg Glu AspAsp Asp Asp Leu 835 840 845 Ala Pro Gly Val Asn Glu Met Ile Arg Ile TyrVal Ala Gln Lys Arg 850 855 860 Lys Ile Gln Asp Gly Asp Lys Leu Ala GlyArg His Gly Asn Lys Gly 865 870 875 880 Val Val Gly Lys Ile Leu Pro GlnGlu Asp Met Pro Phe Leu Pro Asp 885 890 895 Gly Thr Pro Val Asp Ile IleLeu Asn Thr His Gly Val Pro Arg Arg 900 905 910 Met Asn Ile Gly Gln ValLeu Glu Thr His Leu Gly Trp Leu Ala Ser 915 920 925 Ala Gly Trp Ser ValAsp Pro Glu Asp Pro Glu Asn Ala Glu Leu Val 930 935 940 Lys Thr Leu ProAla Asp Leu Leu Glu Val Pro Ala Gly Ser Leu Thr 945 950 955 960 Ala ThrPro Val Phe Asp Gly Ala Ser Asn Glu Glu Leu Ala Gly Leu 965 970 975 LeuAla Asn Ser Arg Pro Asn Arg Asp Gly Asp Val Met Val Asn Ala 980 985 990Asp Gly Lys Ala Thr Leu Ile Asp Gly Arg Ser Gly Glu Pro Tyr Pro 995 10001005 Tyr Pro Val Ser Ile Gly Tyr Met Tyr Met Leu Lys Leu His His 10101015 1020 Leu Val Asp Glu Lys Ile His Ala Arg Ser Thr Gly Pro Tyr Ser1025 1030 1035 Met Ile Thr Gln Gln Pro Leu Gly Gly Lys Ala Gln Phe GlyGly 1040 1045 1050 Gln Arg Phe Gly Glu Met Glu Val Trp Ala Met Gln AlaTyr Gly 1055 1060 1065 Ala Ala Tyr Thr Leu Gln Glu Leu Leu Thr Ile LysSer Asp Asp 1070 1075 1080 Val Val Gly Arg Val Lys Val Tyr Glu Ala IleVal Lys Gly Glu 1085 1090 1095 Asn Ile Pro Asp Pro Gly Ile Pro Glu SerPhe Lys Val Leu Leu 1100 1105 1110 Lys Glu Leu Gln Ser Leu Cys Leu AsnVal Glu Val Leu Ser Ala 1115 1120 1125 Asp Gly Thr Pro Met Glu Leu AlaGly Asp Asp Asp Asp Phe Asp 1130 1135 1140 Gln Ala Gly Ala Ser Leu GlyIle Asn Leu Ser Arg Asp Glu Arg 1145 1150 1155 Ser Asp Ala Asp Thr Ala1160

1. An isolated polynucleotide which encodes a protein comprising theamino acid sequence of SEQ ID NO:
 2. 2. The isolated polynucleotide ofclaim 1, wherein said protein has ribosomal protein S12 activity.
 3. Anisolated polynucleotide, which comprises SEQ ID NO:
 1. 4. An isolatedpolynucleotide which is complimentary to the polynucleotide of claim 3.5. An isolated polynucleotide which is at least 70% identical to thepolynucleotide of claim
 3. 6. An isolated polynucleotide which is atleast 80% identical to the polynucleotide of claim
 3. 7. An isolatedpolynucleotide which is at least 90% identical to the polynucleotide ofclaim
 3. 8. An isolated polynucleotide which hybridizes under stringentconditions to the polynucleotide of claim 3; wherein said stringentconditions comprise washing in 5×SSC at a temperature from 50 to 68° C.9. The isolated polynucleotide of claim 3, which encodes a proteinhaving ribosomal protein S12 activity.
 10. An isolated polynucleotidewhich comprises at least 15 consecutive nucleotides of thepolynucleotide of claim
 3. 11. An isolated polynucleotide which encodesa protein comprising the amino acid sequence of SEQ ID NO:
 4. 12. Anisolated polynucleotide which comprises SEQ ID NO:
 3. 13. A vectorcomprising the isolated polynucleotide of claim
 1. 14. A vectorcomprising the isolated polynucleotide of claim
 3. 15. A vectorcomprising the isolated polynucleotide of claim
 11. 16. A vectorcomprising the isolated polynucleotide of claim
 12. 17. A host cellcomprising the isolated polynucleotide of claim
 1. 18. A host cellcomprising the isolated polynucleotide of claim
 3. 19. A host cellcomprising the isolated polyncleotide of claim
 11. 20. A host cellcomprising the isolated polyncleotide of claim
 12. 21. The host cell ofclaim 17, which is a Coryneform bacterium.
 22. The host cell of claim18, which is a Coryneform bacterium.
 23. The host cell of claim 17,wherein said host cell is selected from the group consisting ofCoryneform glutamicum, Corynebacterium acetoglutamicum, Corynebacteriumthermoaminogenes, Corynebacterium melassecola, Brevibacterium flavum,Brevibacterium lactofermentum, and Brevibacterium divaricatum.
 24. Thehost cell of claim 17, wherein said host cell is selected from the groupconsisting of Corynebacterium glutamicum FERM 1709, Brevibacteriumflavum FERM-P 1708, Brevibacterium.lactofermentum FERM-P1712,Corynebacterium glutamicum FERM-P6463, Corynebacterium glutamicumFERM-P6464, Corynebacterium glutamicum DM58-1, Corynebacteriumglutamicum DG 52-5, Corynebacterium glutamicum DSM 5714 andCorynebacterium glutamicum DSM-12866.
 25. The host cell of claim 18,wherein said host cell is selected from the group consisting ofCoryneform glutamicum, Corynebacterium acetoglutamicum, Corynebacteriumthermoaminogenes, Corynebacterium melassecola, Brevibacterium flavum,Brevibacterium lactofermentum, and Brevibacterium divaricatum.
 26. Thehost cell of claim 18, wherein said host cell is selected from the groupconsisting of Corynebacterium glutamicum FERM 1709, Brevibacteriumflavum FERM-P 1708, Brevibacterium.lactofermentum FERM-P1712,Corynebacterium glutamicum FERM-P6463, Corynebacterium glutamicumFERM-P6464, Corynebacterium glutamicum DM58-1, Corynebacteriumglutamicum DG 52-5, Corynebacterium glutamicum DSM 5714 andCorynebacterium glutamicum DSM-12866.
 27. A Coryneform bacterium whichcomprises an enhanced rpsL gene.
 28. The Coryneform bacterium of claim27, wherein said rpsL gene comprises the polynucleotide sequence of SEQID NO:
 1. 29. The Coryneform bacterium of claim 27, wherein saidenhanced rpsL gene comprises the polynucleotide sequence of SEQ ID NO:3.
 30. Coryneform glutamicum DSM
 1545. 31. A process for producingL-amino acids comprising culturing the host cell of claim 17 in a mediumsuitable for the expression of the polynucleotide; and collecting theL-amino acid.
 32. The process of claim 31, wherein said L-amino acid isL-lysine or L-glutamate.
 33. The process of claim 31, wherein the hostcell further comprises at least one gene whose expression is enhanced,wherein said gene is selected from the group consisting of dapA, gap,tpi, pgk, zwf, pyc, mqo, lys C, lys E, zwa1 and rpoB.
 34. The process ofclaim 31, wherein the host cell further comprises at least one genewhose expression is attenuated, wherein said gene is selected from thegroup consisting of pck gene, pgi gene, poxB, and zwa2.
 35. A processfor producing L-amino acids comprising culturing the host cell of claim18 in a medium suitable for the expression of the polynucleotide; andcollecting the L-amino acid.
 36. The process of claim 35, wherein saidL-amino acid is L-lysine or L-glutamate.
 37. The process of claim 35,wherein the host cell further comprises at least one gene whoseexpression is enhanced, wherein said gene is selected from the groupconsisting of dapA, gap, tpi, pgk, zwf, pyc, mqo, lys C, lys E, zwa1 andrpoB.
 38. The process of claim 35, wherein the host cell furthercomprises at least one gene whose expression is attenuated, wherein saidgene is selected from the group consisting of pck gene, pgi gene, poxB,and zwa2.
 39. A process for producing L-amino acids comprising culturingthe host cell of claim 30 in a medium suitable for the expression of thepolynucleotide; and collecting the L-amino acid.
 40. The process ofclaim 39, wherein said L-amino acid is L-lysine or L-glutamate.
 41. Theprocess of claim 39, wherein the host cell further comprisesat least onegene whose expression is enhanced, wherein said gene is selected fromthe group consisting of dapA, gap, tpi, pgk, zwf, pyc, mqo, lys C, lysE, zwa1 and rpoB.
 42. The process of claim 39, wherein the host cellfurther comprises at least one gene whose expression is attenuated,wherein said gene is selected from the group consisting of pck gene, pgigene, poxB, and zwa2.
 43. A process for producing L-amino acidscomprising culturing the host cell of claim 11 in a medium suitable forthe expression of the polynucleotide; and collecting the L-amino acid.44. The process of claim 43, wherein said L-amino acid is L-lysine orL-glutamate.
 45. The process of claim 43, wherein the host cell furthercomprisesat least one gene whose expression is enhanced, wherein saidgene is selected from the group consisting of dapA, gap, tpi, pgk, zwf,pyc, mqo, lys C, lys E, zwa1 and rpoB.
 46. The process of claim 43,wherein the host cell further comprises at least one gene whoseexpression is attenuated, wherein said gene is selected from the groupconsisting of pck gene, pgi gene, poxB, and zwa2.
 47. A process forproducing L-amino acids comprising culturing the host cell of claim 12in a medium suitable for the expression of the polynucleotide; andcollecting the L-amino acid.
 48. The process of claim 47, wherein saidL-amino acid is L-lysine or L-glutamate.
 49. The process of claim 47,wherein the host cell further comprisesat least one gene whoseexpression is enhanced, wherein said gene is selected from the groupconsisting of dapA, gap, tpi, pgk, zwf, pyc, mqo, lys C, lys E, zwa1 andrpoB.
 50. The process of claim 47, wherein the host cell furthercomprises at least one gene whose expression is attenuated, wherein saidgene is selected from the group consisting of pck gene, pgi gene, poxB,and zwa2.
 51. A process for screening for polynucleotides which encode aprotein having ribosomal protein S12 activity comprising hybridizing theisolated polynucleotide of claim 1 to the polynucleotide to be screened;expressing the polynucleotide to produce a protein; and detecting thepresence or absence of ribosomal protein S12 activity in said protein.52. A process for screening for polynucleotides which encode a proteinhaving ribosomal protein S12 activity comprising hybridizing theisolated polynucleotide of claim 3 to the polynucleotide to be screened;expressing the polynucleotide to produce a protein; and detecting thepresence or absence of ribosomal protein S12 activity in said protein.53. A process for screening for polynucleotides which encode a proteinhaving ribosomal protein S12 activity comprising hybridizing theisolated polynucleotide of claim 10 to the polynucleotide to bescreened; expressing the polynucleotide to produce a protein; anddetecting the presence or absence ribosomal protein S12 activity in saidprotein.
 54. A method for detecting a nucleic acid with at least 70%homology to nucleotide of claim 1, comprising contacting a nucleic acidsample with a probe or primer comprising at least 15 consecutivenucleotides of the nucleotide sequence of claim 1, or at least 15consecutive nucleotides of the complement thereof.
 55. A method forproducing a nucleic acid with at least 70% homology to nucleotide ofclaim 1, comprising contacting a nucleic acid sample with a primercomprising at least 15 consecutive nucleotides of the nucleotidesequence of claim 1, or at least 15 consecutive nucleotides of thecomplement thereof.
 56. A method for detecting a nucleic acid with atleast 70% homology to nucleotide of claim 3, comprising contacting anucleic acid sample with a probe or primer comprising at least 15consecutive nucleotides of the nucleotide sequence of claim 3, or atleast 15 consecutive nucleotides of the complement thereof.
 57. A methodfor producing a nucleic acid with at least 70% homology to nucleotide ofclaim 3, comprising contacting a nucleic acid sample with a primercomprising at least 15 consecutive nucleotides of the nucleotidesequence of claim 3, or at least 15 consecutive nucleotides of thecomplement thereof.
 58. A method for making ribosomal protein S12,comprising: culturing the host cell of claim 17 for a time and underconditions suitable for expression of ribosomal protein S12, andcollecting the ribosomal protein S12.
 59. A method for making 1ribosomal protein S12, comprising: culturing the host cell of claim 18for a time and under conditions suitable for expression of ribosomalprotein S12, and collecting the ribosomal protein S12.
 60. A method formaking ribosomal protein S12, comprising: culturing the host cell ofclaim 19 for a time and under conditions suitable for expression ofribosomal protein S12, and collecting the ribosomal protein S12.
 61. Amethod for making ribosomal protein S12, comprising: culturing the hostcell of claim 20 for a time and under conditions suitable for expressionof ribosomal protein S12, and collecting the ribosomal protein S12.